Inflammatory bowel disease (IBD), consisting of ulcerative colitis (UC) and Crohn's disease (CD), is a source of extensive morbidity and risk for progression to colon cancer in the US population, with more than one million Americans affected. The semi-essential amino acid L-arginine (L-Arg) has been touted as a supplement with many potential benefits including enhancement of immunity. In response to RFA AT-07-004 we will conduct mechanistic studies in mouse models to address the potential value of L-Arg as a supplement in IBD and determine if it can reduce risk for colitis-associated cancer. During conditions of immune activation, the two major routes for L-Arg utilization are the inducible nitric oxide (NO) synthase (iNOS) and arginase pathways. Arginase generates L-ornithine, the substrate for the enzyme ornithine decarboxylase (ODC) that produces polyamines, which have been shown to enhance wound repair and have been implicated as regulatory factors in immune responses. Our data in two murine models of colitis indicate that supplementation of mice with L-Arg or deletion of iNOS improves colitis, while inhibition of arginase or ODC exacerbates disease. L-Arg uptake into cells is primarily dependent on the cationic amino acid transporter (CAT) proteins, particularly CAT2, which is the predominant transporter in macrophages and CAT1, which is ubiquitously expressed. We will show preliminary data that L-Arg availability is important in cellular functions such as global protein translation, wound repair, and responses to inflammatory stimuli. We will also show that CAT2 expression is upregulated in colitis tissues and colitis-associated tumors, and that mice deficient in CAT2 appear to have exacerbation of colitis. Our hypothesis is that L-Arg availability is an important regulator of mucosal inflammation in IBD such that the amount of L-Arg in the extracellular environment, the transport of L- Arg from outside to inside cells, and the balance of competing metabolic pathways for the utilization of L-Arg are all important in immunomodulation. Our specific aims are to determine the biological effects of L-Arg supplementation and the role of its uptake and metabolism in models in which we will study: 1.) In vitro: A.) epithelial integrity;and B.) macrophage function;2.) In vivo colitis: A.) clinical parameters;B.) tissue iNOS and polyamine levels, and serum NO and L-Arg levels;C.) biological effects on colonic epithelial cells and macrophages, D.) responses in colonic lymphocytes;3.) In vivo colitis-associated cancer: A.) clinical parameters;and B.) tumor CAT1, CAT2, iNOS, cytokine, and polyamine levels, and serum NO and L-Arg levels. In these studies, we seek to establish new insights into the role of L-Arg in mucosal immunology and to provide evidence for the potential beneficial effects of L-Arg supplementation in both IBD and IBD-associated cancer while establishing the importance of L-Arg uptake in this process. This work is expected to provide the groundwork for translational studies in patients that could lead to new adjunctive therapies that would be safe, effective, and low in cost and improve the long-term outcome in IBD. PUBLIC HEALTH REVELANCE: Inflammatory bowel disease (IBD) affects more than one million Americans and results in a substantial amount of suffering and the risk for developing cancer of the colon. In this project we will test the hypothesis that supplementation of the amino acid L-arginine (L-Arg) is beneficial in these diseases and that L-Arg uptake into cells has an essential role in the modulation of intestinal inflammation. We will use in vitro and in vivo models of colitis and colitis-associated cancer to conduct mechanistic studies that are designed to generate new insights into the immunobiology of IBD and lay the groundwork for new approaches to attenuate inflammation and associated carcinogenesis.